CN102703411B - Aramagnetic epoxy group mesoporous molecular sieve for immobilized biological enzymes, and preparation method thereof - Google Patents
Aramagnetic epoxy group mesoporous molecular sieve for immobilized biological enzymes, and preparation method thereof Download PDFInfo
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- CN102703411B CN102703411B CN201210184719.6A CN201210184719A CN102703411B CN 102703411 B CN102703411 B CN 102703411B CN 201210184719 A CN201210184719 A CN 201210184719A CN 102703411 B CN102703411 B CN 102703411B
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- 239000002808 molecular sieve Substances 0.000 title claims abstract description 104
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 title claims abstract description 104
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 35
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 125000003700 epoxy group Chemical group 0.000 title abstract description 5
- 108010093096 Immobilized Enzymes Proteins 0.000 claims abstract description 62
- 239000002105 nanoparticle Substances 0.000 claims abstract description 28
- 230000005291 magnetic effect Effects 0.000 claims abstract description 25
- 239000002245 particle Substances 0.000 claims abstract description 7
- 239000004593 Epoxy Substances 0.000 claims description 53
- 230000005298 paramagnetic effect Effects 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 26
- 230000005408 paramagnetism Effects 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 14
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 claims description 10
- 229910052760 oxygen Inorganic materials 0.000 claims description 10
- 239000001301 oxygen Substances 0.000 claims description 10
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 10
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 229920002415 Pluronic P-123 Polymers 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 238000001291 vacuum drying Methods 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 5
- 238000010792 warming Methods 0.000 claims description 5
- 239000007900 aqueous suspension Substances 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
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- 238000010276 construction Methods 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- LDDQLRUQCUTJBB-UHFFFAOYSA-N ammonium fluoride Chemical compound [NH4+].[F-] LDDQLRUQCUTJBB-UHFFFAOYSA-N 0.000 claims description 2
- 238000000975 co-precipitation Methods 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 230000002045 lasting effect Effects 0.000 claims description 2
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims 3
- 230000000694 effects Effects 0.000 abstract description 35
- 108010073038 Penicillin Amidase Proteins 0.000 abstract description 23
- 239000011148 porous material Substances 0.000 abstract description 13
- 230000000977 initiatory effect Effects 0.000 abstract description 12
- 230000000717 retained effect Effects 0.000 abstract description 11
- 238000000926 separation method Methods 0.000 abstract description 9
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- 239000004382 Amylase Substances 0.000 abstract description 2
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- 108010065511 Amylases Proteins 0.000 abstract description 2
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- BPSIOYPQMFLKFR-UHFFFAOYSA-N trimethoxy-[3-(oxiran-2-ylmethoxy)propyl]silane Chemical compound CO[Si](OC)(OC)CCCOCC1CO1 BPSIOYPQMFLKFR-UHFFFAOYSA-N 0.000 abstract 1
- 239000012588 trypsin Substances 0.000 abstract 1
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- NGHVIOIJCVXTGV-UHFFFAOYSA-N 6beta-amino-penicillanic acid Natural products OC(=O)C1C(C)(C)SC2C(N)C(=O)N21 NGHVIOIJCVXTGV-UHFFFAOYSA-N 0.000 description 4
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- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 4
- NVIAYEIXYQCDAN-CLZZGJSISA-N 7beta-aminodeacetoxycephalosporanic acid Chemical compound S1CC(C)=C(C(O)=O)N2C(=O)[C@@H](N)[C@@H]12 NVIAYEIXYQCDAN-CLZZGJSISA-N 0.000 description 3
- 108010029541 Laccase Proteins 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
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- IYNDLOXRXUOGIU-LQDWTQKMSA-M benzylpenicillin potassium Chemical class [K+].N([C@H]1[C@H]2SC([C@@H](N2C1=O)C([O-])=O)(C)C)C(=O)CC1=CC=CC=C1 IYNDLOXRXUOGIU-LQDWTQKMSA-M 0.000 description 2
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- 230000035484 reaction time Effects 0.000 description 2
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- NWXMGUDVXFXRIG-WESIUVDSSA-N (4s,4as,5as,6s,12ar)-4-(dimethylamino)-1,6,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4,4a,5,5a-tetrahydrotetracene-2-carboxamide Chemical compound C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(=O)C(C(N)=O)=C(O)[C@@]4(O)C(=O)C3=C(O)C2=C1O NWXMGUDVXFXRIG-WESIUVDSSA-N 0.000 description 1
- WNQJZQMIEZWFIN-UHFFFAOYSA-N 1-(benzenesulfonyl)-4-(2-chlorobenzoyl)piperazine Chemical compound ClC1=CC=CC=C1C(=O)N1CCN(S(=O)(=O)C=2C=CC=CC=2)CC1 WNQJZQMIEZWFIN-UHFFFAOYSA-N 0.000 description 1
- MWRWFPQBGSZWNV-UHFFFAOYSA-N Dinitrosopentamethylenetetramine Chemical compound C1N2CN(N=O)CN1CN(N=O)C2 MWRWFPQBGSZWNV-UHFFFAOYSA-N 0.000 description 1
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- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 description 1
- 229960003022 amoxicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
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- 229960004841 cefadroxil Drugs 0.000 description 1
- NBFNMSULHIODTC-CYJZLJNKSA-N cefadroxil monohydrate Chemical compound O.C1([C@@H](N)C(=O)N[C@H]2[C@@H]3N(C2=O)C(=C(CS3)C)C(O)=O)=CC=C(O)C=C1 NBFNMSULHIODTC-CYJZLJNKSA-N 0.000 description 1
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- ZAIPMKNFIOOWCQ-UEKVPHQBSA-N cephalexin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@@H]3N(C2=O)C(=C(CS3)C)C(O)=O)=CC=CC=C1 ZAIPMKNFIOOWCQ-UEKVPHQBSA-N 0.000 description 1
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- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 description 1
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention belongs to the technical field of biology, and discloses an aramagnetic epoxy group mesoporous molecular sieve for immobilizing biological enzymes, and a preparation method thereof. According to the invention, gamma glycidoxy propyl trimethoxy silane is utilized to introduce aramagnetic epoxy groups onto the surface of the mesoporous molecular sieve, and then a covalent bond is utilized to immobilize aramagnetic Fe3O4 nano particles and biological enzymes onto the outer surface and the inner surface of the mesoporous molecular sieve respectively, wherein the aramagnetic Fe3O4 nano particles are subjected to L-cysteine surface modification and have the particle sizes larger than the pore diameter of the mesoporous molecular sieve, as a result, specially-structured aramagnetic immobilized enzymes can be prepared and are separated from the liquid phase system easily under the action of an external magnetic field, and further, the property and separation efficiency of the immobilized enzymes are improved. The aramagnetic epoxy group mesoporous molecular sieve can be used for immobilization of water-soluble biological enzymes such as penicillin acylase, glucose isomerase, glucosylase, trypsin and amylase, the activity of the prepared aramagnetic immobilized penicillin acylase is 8800 U/g, and 94.5% of the initial activity is retained after ten times of cycle use.
Description
Technical field
The present invention relates to a kind of paramagnetic epoxy mesoporous molecular sieve for bio-enzyme immobilization and preparation method thereof, belong to biological technical field.
Background technology
Penicillin acylase (EC 3.5.1.11, the enzyme molecular dimension:
) be the enzyme of most critical during semi-synthetic β-lactam antibitics is produced, it can catalysis penicillin and the ring expansion acid hydrolysis remove side chain, produce important intermediate 6-amino-penicillanic acid (6-APA) and the 7-ADCA (7-ADCA) of semi-synthetic β-lactam antibitics, again can catalysis 6-APA and 7-ADCA and side chain condensation, produce multiple semi-synthetic β-lactam antibitics (as Ampicillin, Amoxicillin, Cephalexin and Cefadroxil etc.).
Resolvase is directly used in to catalytic process and has many deficiencies, as unstable in high temperature, strong acid, highly basic and organic solvent, easy loss of catalytic activity; Resolvase reclaims difficulty, unreasonable economically, also causes product to be difficult to separating-purifying, has a strong impact on quality product; Production process is difficult to realize operate continuously, can only disposable periodical operation etc.Immobilized enzyme has overcome the above-mentioned deficiency of resolvase, has not only kept the distinctive catalysis characteristics of resolvase, has also improved operational stability, production process is easy to realize operate continuously, be easy to product separation after having reacted and can reuse, the product purity of gained is high, and production cost is low.Therefore, the immobilization of enzyme is the study hotspot in the fields such as catalytic chemistry, biological chemistry and materials chemistry always.
The bio-enzyme immobilization carrier can be divided into two classes: inorganic carrier and organic carrier.With widely used organic carrier, compare, inorganic carrier has higher physical strength and chemical stability preferably, its structure and surface properties are easily controlled, its outstanding advantage is that the enzyme of load just can be removed through simple process such as roastings, carrier can be reused, this just greatly reduces the cost of immobilized enzyme, has also avoided the aftertreatment problem of the immobilized enzyme of inactivation, has alleviated the pressure to environment.New Mesoporous Molecular Sieves has larger continuously adjustable aperture, high specific surface area, be rich in the slightly acidic hydroxyl in larger loading capacity and duct, can make enzyme molecule that volume is larger be fixed in the mesoporous neutralization reaction product of molecular sieve and diffuse out in time duct, keep the suitable microenvironment of immobilized enzyme, thereby the immobilized enzyme made has higher catalytic activity, the use temperature of immobilized enzyme is lower simultaneously, can avoid the poor problem of the ubiquitous hydrothermal stability of mesopore molecular sieve, therefore, mesopore molecular sieve is the rising enzyme immobilization novel inorganic carrier of a class.
We study discovery (Micropor.Mesopor.Mater., 2008,114 (1-3): 507-510; J Mol.Catal.B-Enzym., 2004,30 (2): 75-81), the performance of immobilized penicillin acylated enzyme and the structure of mesopore molecular sieve (crystalline phase, aperture, pore volume and specific surface area) are closely related.The aperture of mesopore molecular sieve is the key factor that affects activity of the immobilized enzyme; when the aperture of mesopore molecular sieve is greater than the molecular dimension of penicillin acylase; in the immobilization process of enzyme; the enzyme molecule just easily enters in the mesopore molecular sieve duct and combines with surperficial functional groups; take full advantage of the pore volume of mesopore molecular sieve, the immobilized enzyme obtained has greater activity.The crystalline phase of mesopore molecular sieve, pore volume and specific surface area have considerable influence to activity of the immobilized enzyme.SBA-15 mesopore molecular sieve and MCM-41 mesopore molecular sieve (activity of the immobilized enzyme is 402U/g) are similar, are all the one-dimensional tunnel structure of six side's phase p6mm, take the immobilized enzyme poor-performing (activity of the immobilized enzyme is 1343U/g) that it prepares as carrier; And the KIT-6 mesopore molecular sieve with Emission in Cubic Ia3d structure of larger aperture has and the similar three-dimensional open-framework of specific surface area, pore volume and co-continuous of MCM-48 mesopore molecular sieve (activity of the immobilized enzyme is 1509U/g), but the former has larger aperture than the latter, diffusion to enzyme, substrate and product molecule has excellent transmission performance, and the immobilized enzyme that the former prepares as carrier of take has higher activity (activity of the immobilized enzyme is 3522U/g).Spumescence mesopore molecular sieve (Mesostructured Cellular Foams, MCFs) (J.Am.Chem.Soc., 1999,121 (1): 254-255), it is a kind of mesopore silicon oxide with ultra-large aperture and 3D foamy structure, its spherical pore chamber becomes three-dimensional open-framework by the window connection of homogeneous, and aperture is 16～42nm, and pore volume is 1.0～2.4cm
3/ g, owing to having larger window diameter and spherical pore chamber, the transmission that makes more enzyme molecule can enter spherical pore chamber interior and substrate and product is more prone to.We study discovery, and the activity of immobilized penicillin acylated enzyme prepared as carrier by the MCFs mesopore molecular sieve of take can reach 9104U/g.Therefore, the MCFs mesopore molecular sieve is expected to become the enzyme immobilization inorganic carrier of excellent performance.
The mesopore molecular sieve particle diameter is less, more is conducive to the activity of penicillin acylase at the immobilization on mesopore molecular sieve surface and raising immobilized enzyme, but causes immobilized enzyme to be difficult to effectively be separated in reusing process.Owing to normally combining with weak Hyarogen-bonding between the surperficial slightly acidic hydroxyl of penicillin acylase molecule and mesopore molecular sieve, in use a part of enzyme can come off, and operational stability remains further to be improved.Covalent coupling method is, by covalent linkage, the functional groups of the nonessential side-chain radical of the activity of enzyme and fixation support is carried out to the method that coupling prepares immobilized enzyme, because of between enzyme and carrier, with covalent linkage, combine, presenting good operational stability, is current industrial widely used enzyme immobilization method.Therefore, the present invention will be by MCFs mesopore molecular sieve and the paramagnetic Fe of surface graft epoxy functionality
3o
4nanoparticle effectively, in conjunction with preparing paramagnetic epoxy mesoporous molecular sieve fixation support, can be separated rapidly immobilized enzyme under the effect of externally-applied magnetic field, improves the separation efficiency of immobilized enzyme.
The magnetic mesoporous molecular sieve of bibliographical information is all with paramagnetic Fe at present
3o
4or Fe
3o
4@SiO
2for core, the then magnetic mesoporous molecular sieve of synthetic kernel shell structure (Adv.Funct.Mater., 2004,14 (4): 345-351 under alkaline condition; J.Am.Chem.Soc., 2005,127 (25): 8916-8917; J.Am.Chem.Soc., 2006,128 (22): 7130-7131; J.Am.Chem.Soc., 2008,130 (1): 28-29).And the MCFs mesopore molecular sieve under strong acidic condition, take the P123 nonionogenic tenside as template synthetic, Fe
3o
4easily by acid etching and P123, be difficult to be adsorbed on Fe under strong acidic condition
3o
4@SiO
2surface, just can't prepare paramagnetic MCFs mesopore molecular sieve with conventional magnetic mesoporous molecular sieve preparation method like this.The people such as Lu Anhui (J.Am.Chem.Soc., 2004,126 (28): 8616-8617) take the hole-blocking agent of polymethylmethacrylate as the SBA-15 mesopore molecular sieve, then will coat the outside surface of the paramagnetism Co nanoparticle deposition of carbon-coating at molecular sieve, then remove hole-blocking agent with 850 ℃ of high-temperature roastings, prepared paramagnetic SBA-15 mesopore molecular sieve, but this preparation process is comparatively loaded down with trivial details.
Chinese patent CN102286455A discloses a kind of immobilization laccase and preparation method thereof.This material is to take magnetic mesoporous carbon as carrier, laccase is fixed on magnetic mesoporous carbon by physisorption, the duct of magnetic mesoporous carbon is embedded in magnetic nanoparticle, laccase adsorptive capacity on magnetic mesoporous carbon is more than 140mg/g, activity recovery is 60%～95%, and the material aperture is 4-18nm, saturation magnetisation value lower (4.1emu/g), the magnetic separating power is more weak, so that disengaging time is long.
Chinese patent CN101256864A discloses a kind of Superparamagnetism mesoporous silicon dioxide complex microsphere and preparation method thereof.The kernel of this complex microsphere is magnetic ferrite nanoparticle cluster, the spheroid of the coating mesoporous silicon-dioxide of shell, and in particulate, the quality percentage composition of ferrite nano particles is 40～80%.This material has larger specific surface area (300-1000m
2/ g) and stronger magnetic separating power (20-80emu/g), good dispersity in water, surface is a modified laggard one-step functional easily, but the maximum diameter of hole of this material only has 3.8nm, and a lot of macromole enzymes are difficult to enter duct, and adsorptive capacity is lower.
Chinese patent CN101752048A discloses a kind of orderly short duct magnetic mesoporous material.This mesoporous material in the alkaline system of traditional synthesize meso-porous material, add surface to be coated with silicon oxide with Fe
3o
4for the nano magnetic particle of magnetic kernel and by the pH value of conditioned reaction solution, prepared a kind of ordered short-channel and pattern magnetic mesoporous material clearly, may be used in bioseparation, pharmaceutical carrier and catalyzed reaction, but the aperture of this material is smaller, be about 3nm, be difficult to the macromolecular enzyme of absorption it is entered in duct.
Chinese patent CN102389771A discloses a kind of preparation method of bell type magnetic mesoporous silica-microsphere sorbent material.By the selective etch process, utilize mesoporous silicon by macro nanometer γ-Fe
2o
3particle coats, and obtains the magnetic mesoporous silicon microballoon of hollow matrix material, and as tsiklomitsin and 2 kinds of microbiotic of sulphamethazine in sorbent material sharp separation aqueous systems, this synthetic material aperture is about 3nm, is not suitable for equally the absorption of macromole enzyme.
Summary of the invention
The object of the invention is exactly to provide a kind of preparation method who improves the paramagnetic epoxy mesoporous molecular sieve for bio-enzyme immobilization of immobilized enzyme performance and separation efficiency in order to overcome the defect that above-mentioned prior art exists.
Purpose of the present invention can be achieved through the following technical solutions: first use γ-[(2,3)-epoxy the third oxygen] propyl trimethoxy silicane introduces epoxy functionality in the interior appearance of mesopore molecular sieve, do not need further activation just can use the covalent attachment mode will be through the paramagnetism Fe of Cys finishing
3o
4nanoparticle (Fe
3o
4particle diameter is greater than the aperture of mesopore molecular sieve, is difficult to enter in the duct of mesopore molecular sieve) the grafting internal surface that is fixed on mesopore molecular sieve at outside surface and the biological enzyme of mesopore molecular sieve, thus improve performance and the separation efficiency of immobilized enzyme.
Described γ-[(2,3)-epoxy the third oxygen] propyl trimethoxy silicane is to the functional modification on mesopore molecular sieve surface, by mesopore molecular sieve and γ-[(2,3)-epoxy the third oxygen] propyl trimethoxy silicane adds in toluene solution, 100～120 ℃ of reflux 6～18 hours, then mesopore molecular sieve is filtered with after washing with alcohol, in 60～120 ℃ of vacuum drying ovens, drying is 6～18 hours, obtains epoxy mesoporous molecular sieve.
The mass ratio of described γ-[(2,3)-epoxy the third oxygen] propyl trimethoxy silicane and mesopore molecular sieve is 0.5～2: 1.
Described Cys is to paramagnetism Fe
3o
4the finishing of nanoparticle is by the Fe synthetic by coprecipitation method
3o
4nanoparticle is scattered in water, by dilute hydrochloric acid regulator solution pH value, is 4.0～5.0, then adds Cys solution, and ultrasonic reaction 15～45 minutes separates and removes the aqueous solution with magnet magnetic, obtains the paramagnetism Fe of Cys finishing
3o
4nanoparticle.
Described Cys and paramagnetism Fe
3o
4the mass ratio of nanoparticle is 0.03～0.35: 1.
The preparation method of described paramagnetic epoxy mesoporous molecular sieve is by the paramagnetism Fe of Cys finishing
3o
4the aqueous suspension of nanoparticle is added drop-wise in the aqueous suspension of epoxy mesoporous molecular sieve, normal-temperature reaction was separated and is removed the aqueous solution with magnet magnetic after 1～5 hour, then in 40～80 ℃ of vacuum drying ovens dry 6～18 hours, obtain paramagnetic epoxy mesoporous molecular sieve.
The paramagnetism Fe of described Cys finishing
3o
4the mass ratio of nanoparticle and epoxy mesoporous molecular sieve is 0.2～0.7: 1.
The mean pore size of the paramagnetic epoxy mesoporous molecular sieve prepared with aforesaid method is 15～30nm, and specific surface area is 350～500m
2/ g, pore volume is at 1.0～2.0cm
3/ g, saturation magnetization is 10～25emu/g, can be used for the immobilization of the water-soluble biological enzymes such as penicillin acylase, glucose isomerase, glucose transglucosidase, trypsinase and amylase, is specially adapted to the immobilization of penicillin acylase.
Compared with prior art, the preparation method's of paramagnetism epoxy mesoporous molecular sieve of the present invention remarkable advantage is, by being greater than through the particle diameter of finishing the paramagnetism Fe in mesopore molecular sieve aperture
3o
4nanometer particle load is to the outside surface of epoxy mesoporous molecular sieve, reduce the obstruction of paramagnetism nanoparticle for the mesopore molecular sieve duct as far as possible, the impact of reduction on aperture, specific surface area and the pore volume of mesopore molecular sieve, do not need further activation just can directly make biological enzyme be fixed on the internal surface of mesopore molecular sieve in the covalent attachment mode, the paramagnetism immobilized enzyme for preparing special construction, can make immobilized enzyme under the effect of externally-applied magnetic field, be easy to separate from liquid-phase system, thus performance and the separation efficiency of raising immobilized enzyme.
One of remarkable advantage of paramagnetism epoxy mesoporous molecular sieve of the present invention is; the molecular dimension of the aperture ratio penicillin acylase of this mesopore molecular sieve is much bigger; and there is larger specific surface area and pore volume; be conducive to the immobilization of enzyme molecule and the diffusion of substrate and product molecule, thereby improve the activity of immobilized enzyme.
Two of the remarkable advantage of paramagnetism epoxy mesoporous molecular sieve of the present invention is, this mesopore molecular sieve, will be through the paramagnetism Fe of Cys finishing by the covalent attachment mode by surperficial epoxide group
3o
4the internal surface that the nanoparticle grafting is fixed on mesopore molecular sieve at outside surface and the biological enzyme of mesopore molecular sieve, thus the operational stability of immobilized enzyme improved.
Three of the remarkable advantage of paramagnetism epoxy mesoporous molecular sieve of the present invention is, this mesopore molecular sieve has paramagnetism, can be under the effect of externally-applied magnetic field, be easy to separate from liquid-phase system, when removing externally-applied magnetic field, immobilized enzyme can be dispersed in liquid-phase system again again, easy and simple to handle, easily industrialization.
The accompanying drawing explanation
The immobilization schematic diagram of the preparation process of the paramagnetic epoxy mesoporous molecular sieve that Fig. 1 is special construction and enzyme.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
In following examples, adopt following method to carry out the immobilization of penicillin acylase, and activity and the operational stability of measuring immobilized enzyme:
The immobilization of penicillin acylase: take 0.020g carrier and the 6.0mL penicillin acylase solution (V through the phosphate buffer soln dilution of pH=7.8
buffer/ V
enzyme=9: 1) mix, put into the shaking bath immobilization of 30 ℃ and carry out the magnetic separation after 12 hours, the gained solid carries out determination of activity after repeatedly washing with the phosphate buffer soln of pH=7.8.
The determination of activity of immobilized enzyme (the penicilline g potassium salt hydrolysis prepares 6-APA): at the temperature of 37 ℃, above-mentioned immobilized enzyme is evenly mixed with the penicilline g potassium salt brine solution (with the dilution of 0.1mol/L pH=7.8 phosphate buffer soln) of 20mL 4wt%, then the NaOH solution titration that is 0.1mol/L by concentration, make the pH value of mixing solutions remain on 7.8, record the consumption of NaOH in 10 minutes.Then calculate the activity of immobilized enzyme with following formula:
A(U/g)＝V
NaOH×C
NaOH×10
3/(m×r)
Wherein A represents the activity of immobilized enzyme; V
naOHrepresent NaOH consumption (ml); C
naOHrepresent NaOH concentration (mol/L); M represents carrier dry weight (g); The t representative test time used (min).
The operational stability of immobilized enzyme is measured: used immobilized enzyme solution is carried out to the magnetic separation, then immobilized enzyme is transferred in reactor, adopt above-mentioned activity determination method to measure the activity of used immobilized enzyme.After 10 times recycle, the activity of immobilized enzyme and the per-cent of initial activity are higher, illustrate that the operational stability of immobilized enzyme is better.
Comparative Examples
At ambient temperature, by 2.0g Pluronic P123 (EO
20pO
70eO
20, M
ay=5800) be dissolved in the HCl solution of 75mL 1.6mol/L; Add 0.023g Neutral ammonium fluoride and 3.0g 1,3 after P123 dissolves fully, the 5-trimethylbenzene, then be warming up to solution 37 ℃ and lasting the stirring after 1 hour, adds the 4.4g tetraethoxy, and under 37 ℃, continue to stir 20 hours; Solution is proceeded to in teflon-lined hydrothermal crystallizing still, in 100 ℃ of hydrothermal crystallizings 24 hours; Filter and obtain white solid after solution is cooled to room temperature, dried overnight in the baking oven of 100 ℃, then be warming up to 500 ℃ of roastings at the retort furnace Program and within 8 hours, obtain mesopore molecular sieve.By 1.0g mesopore molecular sieve and 1.0g γ-[(2,3)-epoxy the third oxygen] propyl trimethoxy silicane joins in the 50mL toluene solution, filter and use washing with alcohol after 12 hours in 110 ℃ of reflux, then the dry epoxy mesoporous molecular sieve that obtains in 12 hours in 90 ℃ of vacuum drying ovens.Immobilization by above-mentioned mesopore molecular sieve for penicillin acylase; the activity of the immobilized enzyme obtained is 9360U/g; after 10 times recycle (with the separation of being fixed of whizzer enzyme, operating process is comparatively complicated), immobilized enzyme has retained 94.6% initial activity.
Embodiment 1
By 0.365g FeCl
36H
2o and 0.208g FeSO
47H
2o (Fe
3+/ Fe
2+mol ratio is 1.8: 1) be dissolved in 10mL water and be warming up to 80 ℃, regulate the pH value with strong aqua and be about 11, at this temperature, ageing is 2 hours, is washed with water to the Fe that neutrality obtains 0.15g after being cooled to room temperature
3o
4nanoparticle, then be scattered in 20mL water, by dilute hydrochloric acid regulator solution pH value, is 4.0～5.0, then add the Cys solution 5mL that concentration is 5g/L, ultrasonic reaction 30 minutes is finally removed the aqueous solution under externally-applied magnetic field, obtains the Fe of Cys finishing
3o
4nanoparticle.
Fe by the Cys finishing of the epoxy mesoporous molecular sieve for preparing in the 0.60g Comparative Examples and 0.15g
3o
4nanoparticle is scattered in respectively in 25mL water, then mixes normal-temperature reaction 5 hours, has reacted with magnet magnetic and has separated and to remove the aqueous solution, then in 60 ℃ of vacuum drying ovens dry 12 hours, obtains paramagnetic epoxy mesoporous molecular sieve.Immobilization by above-mentioned paramagnetic epoxy mesoporous molecular sieve for penicillin acylase; the activity of the immobilized enzyme obtained is 8800U/g; after 10 times recycle (with the separation of being fixed of magnet enzyme, easy and simple to handle), immobilized enzyme has retained 94.5% initial activity.
Embodiment 2
By the Change Weight To 0.70g of epoxy mesoporous molecular sieve in embodiment 1 and the Fe of Cys finishing
3o
4the Change Weight To 0.30g of nanoparticle; other preparation process are identical with embodiment 1; immobilization by the paramagnetic epoxy mesoporous molecular sieve that makes for penicillin acylase; the activity of the immobilized enzyme obtained is 8514U/g; after 10 times recycle, immobilized enzyme has retained 92.8% initial activity.
Embodiment 3
By the Change Weight To 0.45g of epoxy mesoporous molecular sieve in embodiment 1 and the Fe of Cys finishing
3o
4the Change Weight To 0.30g of nanoparticle; other preparation process are identical with embodiment 1; immobilization by the paramagnetic epoxy mesoporous molecular sieve that makes for penicillin acylase; the activity of the immobilized enzyme obtained is 8489U/g; after 10 times recycle, immobilized enzyme has retained 89.8% initial activity.
Embodiment 4
Fe by epoxy mesoporous molecular sieve in embodiment 1 and Cys finishing
3o
4the normal-temperature reaction time of nanoparticle changes 1 hour into; other preparation process are identical with embodiment 1; immobilization by the paramagnetic epoxy mesoporous molecular sieve that makes for penicillin acylase; the activity of the immobilized enzyme obtained is 8950U/g; after 10 times recycle, immobilized enzyme has retained 89.6% initial activity.
Embodiment 5
Fe by epoxy mesoporous molecular sieve in embodiment 1 and Cys finishing
3o
4the normal-temperature reaction time of nanoparticle changes 3 hours into; other preparation process are identical with embodiment 1; immobilization by the paramagnetic epoxy mesoporous molecular sieve that makes for penicillin acylase; the activity of the immobilized enzyme obtained is 8902U/g; after 10 times recycle, immobilized enzyme has retained 92.8% initial activity.
Embodiment 6
Change the concentration of Cys solution in embodiment 1 into 1g/L; other preparation process are identical with embodiment 1; immobilization by the paramagnetic epoxy mesoporous molecular sieve that makes for penicillin acylase; the activity of the immobilized enzyme obtained is 8838U/g; after 10 times recycle, immobilized enzyme has retained 92.3% initial activity.
Embodiment 7
Change the concentration of Cys solution in embodiment 1 into 10g/L; other preparation process are identical with embodiment 1; immobilization by the paramagnetic epoxy mesoporous molecular sieve that makes for penicillin acylase; the activity of the immobilized enzyme obtained is 8893U/g; after 10 times recycle, immobilized enzyme has retained 91.8% initial activity.
Embodiment 8
By the γ in Comparative Examples-[(2; 3)-epoxy the third oxygen] the Change Weight To 0.5g of propyl trimethoxy silicane; other preparation process are identical with embodiment 1 with Comparative Examples; immobilization by the paramagnetic epoxy mesoporous molecular sieve that makes for penicillin acylase; the activity of the immobilized enzyme obtained is 9354U/g; after 10 times recycle, immobilized enzyme has retained 89.1% initial activity.
Embodiment 9
By the γ in Comparative Examples-[(2; 3)-epoxy the third oxygen] the Change Weight To 2.0g of propyl trimethoxy silicane; other preparation process are identical with embodiment 1 with Comparative Examples; immobilization by the paramagnetic epoxy mesoporous molecular sieve that makes for penicillin acylase; the activity of the immobilized enzyme obtained is 8821U/g; after 10 times recycle, immobilized enzyme has retained 92.2% initial activity.
Claims (4)
1. the preparation method for the paramagnetic epoxy mesoporous molecular sieve of bio-enzyme immobilization, it is characterized in that, the method is first used γ-[(2,3)-epoxy the third oxygen] propyl trimethoxy silicane introduces epoxy functionality at the surfaces externally and internally of mesopore molecular sieve, then by the covalent attachment mode, will be greater than through the particle diameter of Cys finishing the paramagnetism Fe in mesopore molecular sieve aperture
3o
4the internal surface that the nanoparticle grafting is fixed on mesopore molecular sieve at outside surface and the biological enzyme of mesopore molecular sieve, prepare the paramagnetism immobilized enzyme of special construction, and concrete operation step is as follows:
Step 1, at ambient temperature, by 2.0g Pluronic P123 (EO
20pO
70eO
20, M
av=5800) be dissolved in the HCl solution of 75mL1.6mol/L; Add 0.023g Neutral ammonium fluoride and 3.0g1 after P123 dissolves fully, 3,5-trimethylbenzene, then be warming up to solution 37 ℃ and lasting the stirring after 1 hour, adds the 4.4g tetraethoxy, and under 37 ℃, continue to stir 20 hours; Solution is proceeded to in teflon-lined hydrothermal crystallizing still, in 100 ℃ of hydrothermal crystallizings 24 hours; Filter and obtain white solid after solution is cooled to room temperature, dried overnight in the baking oven of 100 ℃, then be warming up to 500 ℃ of roastings at the retort furnace Program and within 8 hours, obtain mesopore molecular sieve;
Step 2, by mesopore molecular sieve and γ-[(2,3)-epoxy the third oxygen] propyl trimethoxy silicane adds in toluene solution, 100～120 ℃ of reflux 6～18 hours, then mesopore molecular sieve is filtered with after washing with alcohol, in 60～120 ℃ of vacuum drying ovens, drying is 6～18 hours, obtains epoxy mesoporous molecular sieve;
Step 3, Fe that will be synthetic by coprecipitation method
3o
4nanoparticle is scattered in water, by dilute hydrochloric acid regulator solution pH value, is 4.0～5.0, then adds Cys solution, and ultrasonic reaction 15～45 minutes separates and removes the aqueous solution with magnet magnetic, obtains the paramagnetism Fe of Cys finishing
3o
4nanoparticle;
Step 4, by the paramagnetism Fe of Cys finishing
3o
4the aqueous suspension of nanoparticle is added drop-wise in the aqueous suspension of epoxy mesoporous molecular sieve, normal-temperature reaction was separated and is removed the aqueous solution with magnet magnetic after 1～5 hour, then in 40～80 ℃ of vacuum drying ovens dry 6～18 hours, obtain paramagnetic epoxy mesoporous molecular sieve.
2. the preparation method of the paramagnetic epoxy mesoporous molecular sieve for bio-enzyme immobilization according to claim 1, it is characterized in that, the mass ratio of described γ-[(2,3)-epoxy the third oxygen] propyl trimethoxy silicane and mesopore molecular sieve is 0.5～2: 1.
3. the preparation method of the paramagnetic epoxy mesoporous molecular sieve for bio-enzyme immobilization according to claim 1, is characterized in that, described Cys and paramagnetism Fe
3o
4the mass ratio of nanoparticle is 0.03～0.35: 1.
4. the preparation method of the paramagnetic epoxy mesoporous molecular sieve for bio-enzyme immobilization according to claim 1, is characterized in that, the paramagnetism Fe of described Cys finishing
3o
4the mass ratio of nanoparticle and epoxy mesoporous molecular sieve is 0.2～0.7: 1.
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| CN103012673B (en) * | 2013-01-21 | 2015-03-18 | 华东理工大学 | Biological enzyme immobilizing core-shell type superparamagnetic polymeric microsphere and preparing method thereof |
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